Marijuana is currently the most widely abused street drug. However, the functional significance of the cannabinoid receptor system in health and disease includes the use of cannabinoids as analgesics, antiemetics in cancer patients, anticonvulsants for epilepsy and as antiglaucoma agents as well as immunomodulatory agents. To date, two cannabinoid receptor subtypes, CB1 and CB2, have been fully defined as these two subtypes.. Anandamide produces the full range of behavioral effects (antinociception, catalepsy and impaired locomotor activity) in CB1 receptor knockout mice. In addition, anandamide-stimulated GTPgS activity can be elicited in brain membranes from these mice. Finally, radioligand binding studies indicate the existence of additional binding sites in brain and spinal cord. The goal of this project is to clone and characterize additional subtypes of the cannabinoid receptors in order to elucidate their role in vivo. The first specific aim is to isolate additional cannabinoid receptor subtypes using a cDNA expression library constructed from CB1 receptor knockout mice. Stable cell lines will be used to functionally characterize novel receptor subtypes using GTPgS binding and investigation of signal transduction pathways. The second specific aim will identify additional receptor-subtype selective ligands to establish structure-activity relationships for CB1 and CB2 receptors, in conjunction with other Center grant participants. The second specific aim will identify additional receptor-subtype selective ligands to establish structure-activity relationships for CB1 and CB2 receptors, in conjunction with other Center grant participants. In addition, we will characterize the ligand-receptor-G protein interactions which discriminate between the receptor subtypes. In addition, we will characterize the ligand-receptor-G protein interactions which discriminate between the receptor subtypes. A model is emerging about the specific sites of interaction for ligand recognition and signal transduction in the CB1 and CB2 receptors. The third aim is to define the regions believed to be critical to receptor subtype discrimination by site-directed mutagenesis and construction of receptor chimeras. These studies will facilitate the design of new therapeutic strategies involving the cannabinoid systems. Furthermore, insight into the molecular mechanisms of activation of cannabinoid receptors may lead to a better understanding of marijuana abuse in humans.